SUMMARY OF WORK: Our group is interested in understanding the role of the ShcC adaptor protein in receptor tyrosine kinase (RTK) function in general and neural development in particular. ShcC is a member of the Shc family of adaptor proteins. This family consists of three members (ShcA, ShcB and ShcC), each of which is characterized by the presence of an amino-terminal phosphotyrosine binding domain (PTB), a central Gly- and Pro-rich effector region (CH1), and a carboxy-terminal Src homology 2 (SH2) domain. Although this family of proteins lacks any intrinsic enzymatic activity, Shc proteins act as scaffolds to assemble signaling complexes that regulate the function of RTKs as well as a wide variety of receptor proteins including integrins, G-protein coupled receptors (GPCRs) and hematopoietic receptors. In addition, recent work from other laboratories suggests that certain isoforms of ShcA may play an important role in the response of cells to oxidative stress and in the life span of an organism. In collaboration with Dr. Tony Pawson, we have shown that elimination of ShcC does not affect development of the nervous system suggesting that ShcC may be more important for function rather that development. Using a combination of techniques, we have shown that ShcC expression is restricted to the nervous system and to neurons in particular. To address the importance of ShcC in the nervous system, we have begun to more precisely define both the temporal and spatial expression pattern of ShcC during development. In addition to determining the expression pattern of ShcC, we have begun to experiments to determine the phosphorylation status of ShcC in the whole animal. We have developed phosphospecific antibodies directed against the ?activated? forms of ShcC. Using these antibodies we hope to determine not only where and when ShcC is expressed but also where and when it is activated. In addition, we are utilizing mass spectrometry in order to determine the specific sites of phosphorylation in vivo. These approaches will also prove useful for determining whether environmental insult affects ShcC function. For example, do neurotoxicants affect ShcC expression or tyrosine phosphorylation? To further explore the importance of ShcC, we are using mutant versions of the protein to interfere with RTK signaling. Although ShcC possesses two domains with the potential of interacting with activated RTKs, we have demonstrated that the PTB domain is the predominant means by which ShcC binds to the activated EGFR. Furthermore, our results suggested that the SH2 domain might be important for binding to an additional cellular component(s) necessary for EGFR function. We are currently attempting to isolate SH2 interacting proteins with the hope of identifying important downstream targets of both ShcC and the EGFR.